200 μl/ml salmon sperm DNA and 10nM Cy5 probes. The Bio-strand Tip was washed for 2 min each in 450 μl washing buffer 1 (2xSSC and 0.1% SDS) washing buffer 2 (1xSSC and 0.1 %SDS), washing buffer 3 (0.1xSSC and 0.1% SDS) and then soaked in 450 μl of 2xSSC. The Cy5 probes for ASOCH were: 5’-ATGGCGACCTG 3’ (G), 5’ Cy5-ATGGCAACCTG 3’ (A). Two non-labeled oligonucleotides were used as competitor to the Cy5-probes, (5’-ATGG CAACCTG-3’) for G and (5’-ATGGCGACCTG-3’) for A. The Cy5 fluorescent signal was scanned with the Handy Bio-Strand scanner (PSS, Chiba, Japan) and the results were analyzed with Hy-Soft software (PSS, Chiba, Japan). In addition, all OPRM1 SNP were confirmed by PCR direct sequencing.
